Altered Versican Fibrillar Interactions in the Outflow Pathway

Summary

Our research focuses on the trabecular meshwork (TM), a spongy tissue located in the eye around the base of the cornea, near the ciliary body. The TM is responsible for draining a fluid called aqueous humor from the eye via the anterior chamber, and is believed to be the tissue that regulates intraocular pressure, a critical factor in the development of glaucoma. Our laboratory is investigating the hypothesis that a specific subset of complex molecules that reside in the TM, and that interact with each other, are important in the regulation of aqueous humor outflow resistance. When these processes are disrupted, there will be changes in the tissue that lead to alterations in outflow facility. Thus our primary objective is to improve the overall understanding of how aqueous humor outflow resistance is regulated and to determine the triggers of glaucoma in order to hasten the development of new therapies that help glaucoma patients.

Project Details

Our research focuses on the trabecular meshwork (TM), a discrete tissue located circumferentially around the base of the cornea and near the ciliary body, which is responsible for draining the aqueous humor from the anterior chamber of the eye. The TM is believed to be the tissue that regulates intraocular pressure, a critical factor in the development of glaucoma. The goal of this project is to define how a specific subset of interacting and complex molecules in the TM contribute to the regulation of aqueous humor outflow resistance.

Because the availability of human eye tissue is limited and costly, the experiments in the first and second aims are being performed in primary cell cultures. TM cells can be grown in primary culture and used to evaluate the effects of targeted gene silencing. The objective in the first specific aim is to measure gene-specific changes in primary cultured TM cells in response to gene silencing of versican and fibrillin-1 using quantitative polymerase chain reaction (PCR) arrays. This approach allows us to study the consequences of silencing specific genes involved in outflow resistance on a genetic level. The objective of the second specific aim is identifying proteins altered in response to targeted gene silencing of versican and fibrillin-1 in primary cultured human TM cells. Proteins secreted from untreated and gene-silenced TM cells in culture will be compared and quantitated. This approach allows us to study the consequences of silencing specific genes involved in outflow resistance on a protein level. Finally, the objective in the third specific aim is determining structural changes in TM tissue after versican or fibrillin-1 gene silencing. In this aim we will use an in vitro organ culture model system in which outflow rates are measured before and after gene silencing. Untreated and gene-silenced tissues will then be imaged by confocal microscopy and super-resolution microscopy to assess differences in ultrastructure as a result of targeted gene silencing.

This proposal is innovative in that it uses targeted gene silencing in cultured cells and quantitative PCR arrays, combined with protein identification, to provide new insight into the events that may determine outflow resistance mechanisms. Additionally, anterior segment organ culture is unique in that it is the only ex vivo model system available to test for outflow facility in response to targeted gene silencing. Our primary objective is to improve the overall understanding of how aqueous humor outflow resistance is regulated and what are the triggers in the development of glaucoma. Once completed, the knowledge gained from this study will facilitate the development of novel therapies aimed at helping glaucoma patients.